Background: In recent years, in order to prevent the excessive use of antibiotics, researchers are paying more attention to nanoparticles with antibacterial properties. The aim of this study was to evaluate the antibacterial activity of Zinc Oxide nanoparticles (ZnO-NPs) synthesized by the green method from the extract of the Hyssopus officinail against some of the gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative (Pseudomonas aeruginosa, Escherichia coli).Materials and Methods: For this experimental study, dry powder was prepared from Hyssopus officinail and Zinc acetate was used as a Zinc source. After synthesizing the nanoparticle via green method according to the protocol, concentrations of 250, 500, 1000 and 2000 µg/ml were prepared. The blanc discs with above mentioned concetrations were placed on the plate of bacteria cultured on Muller Hinton Agar culture media. Antibiotic disc gentamicin was used as control.Results: The results showed that ZnO-NPs synthesized from the Hyssopus officinail extract at concentrations of 1000 and 2000 μg/ml had a proper antimicrobial activity, so that the inhibition in the concentration of 2000 μg/ml in E. coli and S. aureus were almost equal to the gentamicin.Conclusion: The results obtained from antibacterial property of ZnO-NPs synthesized from the Hyssopus officinail extract showed that there is a direct correlation between the concentration of nanoparticles and the elimination of bacteria